Vaccinia virus has a genome of nearly 2000,000 base pairs that encodes approximately 200 polypeptides. These genes are expressed within the cytoplasm in a coordinated fashion, so that some polypeptides are made before and other after DNA replication. Enzymes and factors needed for early transcription are packaged within the infectious particle, while those needed for intermediate and late transcription are present in the cytoplasm of infected cells. Vaccinia virus provides and excellent system for combining biochemical and genetic approaches to the study of transcription. The aim of the project is to determine the mechanism regulating gene expression. This past year, progress has been made in defining the viral proteins involved in transcription and in understanding their roles. Emphasis has been on the virus-encoded RNA polymerase and transcription factors. This, three additional RNA polymerase subunit genes encoding polypeptides of 132,000, 35,000, and 30,000 Daltons were identified within the vaccinia virus genome and sequenced. All three genes are expressed both early and late in infection and the protein subunits are assembled into the RNA polymerase complex that is packaged in virions. Interestingly, the 30,000 Dalton subunit is homologous to an eukaryotic transcription elongation factor. One of the factors required for late transcription was shown to be encoded by an intermediate gene consistent with the cascade model of gene regulation. The promoter DNA contacts made by VETF, the vaccinia virus early transcription factor, were determined and evidence was obtained that this interaction leads to bending of the DNA.